Downloaded from www.microbiologyresearch.org by IP: 54.242.161.225 On: Wed, 06 Apr 2016 06:27:38 Bacillus kochii sp. nov., isolated from foods and a pharmaceuticals manufacturing site Herbert Seiler, 1 Verena Schmidt, 1 Mareike Wenning 1 and Siegfried Scherer 1,2 Correspondence Herbert Seiler herbert.seiler@wzw.tum.de 1 Department of Microbiology (ZIEL), Technische Universita ¨t Mu ¨ nchen, Weihenstephaner Berg 3, D-85350 Freising, Germany 2 Lehrstuhl fu ¨ r Mikrobielle O ¨ kologie, Technische Universita ¨t Mu ¨ nchen, Weihenstephaner Berg 3, D-85350 Freising, Germany Three Gram-staining-positive, strictly aerobic, motile, catalase-positive, endospore-forming rods, designated WCC 4582 T , WCC 4581 and WCC 4583, were isolated from two different food sources and a pharmaceuticals production site. The three isolates were highly similar in their 16S rRNA gene sequences (100 % similarity) and groEL sequences (99.2–100 % similarity), Fourier- transform infrared spectroscopic fingerprints and other features tested. The isolates were most closely related to Bacillus horneckiae; the isolates and the type strain of B. horneckiae shared 97.6 % and 89.6 % 16S rRNA gene and groEL sequence similarities, respectively. The organisms grew optimally at 30 6C, at pH 7 and in the presence of 0.5 % (w/v) NaCl. The cell-wall peptidoglycan of WCC 4582 T contained meso-diaminopimelic acid (A1c) and the genomic DNA G+C content was 36.4 mol%. DNA–DNA relatedness between strain WCC 4582 T and B. horneckiae NRRL B-59162 T was 17 %. The three isolates are considered to represent a novel species of the genus Bacillus, for which the name Bacillus kochii sp. nov. is proposed. The type strain is WCC 4582 T (5DSM 23667 T 5CCUG 59877 T 5LMG 25855 T ). Microbial spoilage in food and contamination of phar- maceutical preparations pose a health risk to consumers and cause considerable economic losses in the industry. In particular, spore-forming bacteria are of importance, as they are heat resistant in food and endure desiccation and treatment with inorganic disinfectants, organic solvents and UV light in clean rooms. Formerly, the identification of spoilage micro-organisms was very costly and burdensome. Several research projects, therefore, developed an identifica- tion technology based on Fourier-transform infrared (FT-IR) spectroscopy, which fulfilled the following criteria: ease of use, fast and reliable results, high sample throughput and low costs as no consumables are required. During the course of many studies in our laboratory, thousands of micro-organisms have been isolated from industrial environments and identified by this method and novel species have been discovered (Bleicher et al., 2010; Rieser et al., 2012; Schmidt et al., 2009, 2012). Strains WCC 4582 T and WCC 4583 were isolated from products of two dairies in south Germany and strain WCC 4581 was isolated from a clean room in a pharmaceuticals manufacturing site in north Germany. Isolation from the dairy products was carried out using tryptone soy agar (TSA; Roth) supplemented with 0.1 % glucose (TSGA) or tryptone soy broth (TSB; Roth) solidified with 15 g agar l 21 (TSBA); isolation from pharmaceutical clean room samples was carried out using TSA. For identification purposes, the FT-IR spectra of endospore-forming isolates were recorded as described by Ku ¨mmerle et al. (1998), Oberreuter et al. (2002) and Wenning et al. (2008) using an IFS-28B spectrometer and OPUS version 5.5 (Bruker Optics). The sample spectra were compared with a reference database for aerobic, mesophilic, spore-forming bacteria, including 770 spectra from 108 species and 12 genera. The three isolates described here displayed highly similar spectra but did not match any reference spectrum. The isolates were maintained as glycerol suspensions at 280 u C and as Weihenstephan culture collection (WCC) lyophilized cells. Almost-complete 16S rRNA gene sequences of the isolates (each 1511 bp) were obtained according to Schmidt et al. (2009). The identification of phylogenetic neighbours was initially performed using BLAST, MEGABLAST and FASTA (Altschul et al., 1997; Pearson & Lipman, 1988; Zhang et al., 2000). Searches of public databases indicated that members of a presumably hitherto-unknown species of the Abbreviation: FT-IR, Fourier-transform infrared. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains WCC 4582 T , WCC 4581 and WCC 4583 are FN995265, FR845720 and FR845721, respectively. Three supplementary figures are available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1092–1097 DOI 10.1099/ijs.0.027771-0 1092 027771 G 2012 IUMS Printed in Great Britain